p53 is a critical tumor suppressor in mice and humans, and is the most commonly mutated gene in human cancer. The main goal of this proposal is to understand the molecular mechanisms by which p53 mediates tumor suppression. p53 is able to sense cellular stress and respond by inducing cell cycle arrest, senescence, or apoptosis by activating expression of target genes. Some p53 target genes have known roles in mediating effector functions such as arrest or apoptosis. However the genes important for mediating tumor suppression are unknown. To identify novel p53 target genes involved in tumor suppression, we have used microarray analysis of cells expressing p53 in which residues within each transactivation domain are mutated. Mutation of the first transactivation domain of p53 (p5325,26) severely compromises the ability of p53 to activate gene expression. This mutant is unable to induce expression of most known p53 targets. However, the ability to suppress tumor formation in vivo is retained. These data indicate that the target genes important in mediating tumor suppression have not yet been defined, but p5325,26 can be used to identify candidate genes. In this project we will focus on candidate genes identified using microarray studies to compare gene expression in murine embryonic fibroblasts (MEFs) expressing p53 mutants that can function in tumor suppression (p5325,26, p5353,54, p53wt) with p53 mutants that are unable to suppress tumor formation (p5325,26,53,54 and p53-null). The candidates were further filtered for genes known to be downregulated in a variety of human and mouse tumors, to arrive at a list of 14 candidate genes. In the experiments in this proposal, we will assess both the requirement and sufficiency for candidate genes to mediate oncogene- induced p53 effector function. For these experiments we will utilize two cell culture systems. Both HrasV12 MEFs, the cells in which the candidate genes were originally identified, and a murine lung cancer cell line expressing oncogenic Kras and containing homozygous inducible p53 alleles will be used. In these cells we will express each candidate in the absence of p53 or use shRNAs to knockdown expression of each candidate in the presence of p53 before performing proliferation, senescence, transformation, and migration assays. Further, we will use the shRNA knockdown approach to analyze the importance of the candidates in an in vivo mouse model of non-small cell lung cancer. The experiments proposed in each aim will allow us to determine which candidates are important for the ability of p53 to mediate tumor suppression. If necessary, these experiments can easily be expanded to include additional candidates, or to analyze candidate genes in combination. Understanding the mechanisms that underlie the tumor suppressor function of p53 will have broad implications in tumor cell biology and in development of better cancer treatments. PUBLIC HEALTH RELEVANCE: p53 is an important tumor suppressor protein, mutated in over half of all human cancers, yet the activities of p53 that are important in preventing cancer are unknown. In this project we will identify the molecular mechanisms of tumor suppression by p53. The results of these studies will have broad implications in both biology and medicine, and may aid in the design and development of better cancer therapies.